Neutrophils are the most abundant and shortest-lived leukocytes in humans and tight regulation of neutrophil turnover
constitutive apoptosis is essential for control of infection and resolution of ...inflammation. Accordingly, aberrant neutrophil turnover is hallmark of many disease states. We have shown in previous work that the intracellular bacterial pathogen
markedly prolongs human neutrophil lifespan. This is achieved, in part, by changes in neutrophil gene expression. Still unknown is the contribution of major neutrophil pro-survival signaling cascades to this process. The objective of this study was to interrogate the contributions of ERK and p38 MAP kinase, Class I phosphoinositide 3-kinases (PI3K), AKT, and NF-κB to neutrophil survival in our system. We demonstrate that both ERK2 and p38α were activated in
-infected neutrophils, but only p38α MAPK was required for delayed apoptosis and the rate of cell death in the absence of infection was unchanged. Apoptosis of both infected and uninfected neutrophils was markedly accelerated by the pan-PI3K inhibitor LY2094002, but AKT phosphorylation was not induced, and neutrophil death was not enhanced by AKT inhibitors. In addition, isoform specific and selective inhibitors revealed a unique role for PI3Kα in neutrophil survival after infection, whereas only simultaneous inhibition of PI3Kα and PI3kδ accelerated death of the uninfected controls. Finally, we show that inhibition of NF-κB triggered rapid death of neutrophil after infection. Thus, we defined roles for p38α, PI3Kα and NF-κB delayed apoptosis of
-infected cells and advanced understanding of Class IA PI3K isoform activity in human neutrophil survival.
Summary
Francisella tularensis in an intracellular bacterial pathogen that causes a potentially lethal disease called tularemia. Studies performed nearly 100 years ago revealed that neutrophil ...accumulation in infected tissues correlates directly with the extent of necrotic damage during F. tularensis infection. However, the dynamics and details of bacteria–neutrophil interactions have only recently been studied in detail. Herein, we review current understanding regarding the mechanisms that recruit neutrophils to F. tularensis–infected lungs, opsonization and phagocytosis, evasion and inhibition of neutrophil defense mechanisms, as well as the ability of F. tularensis to prolong neutrophil lifespan. In addition, we discuss distinctive features of the bacterium, including its ability to act at a distance to alter overall neutrophil responsiveness to exogenous stimuli, and the evidence which suggests that macrophages and neutrophils play distinct roles in tularemia pathogenesis, such that macrophages are major vehicles for intracellular growth and dissemination, whereas neutrophils drive tissue destruction by dysregulation of the inflammatory response.
F. novicida acts via the intrinsic and extrinsic pathways to prolong neutrophil viability by a mechanism independent of opsonins and phagocytosis.
Francisella novicida is a Gram‐negative bacterium ...that is closely related to the highly virulent facultative intracellular pathogen, Francisella tularensis. Data published by us and others demonstrate that F. tularensis virulence correlates directly with its ability to impair constitutive apoptosis and extend human neutrophil lifespan. In contrast, F. novicida is attenuated in humans, and the mechanisms that account for this are incompletely defined. Our published data demonstrate that F. novicida binds natural IgG that is present in normal human serum, which in turn, elicits NADPH oxidase activation that does not occur in response to F. tularensis. As it is established that phagocytosis and oxidant production markedly accelerate neutrophil death, we predicted that F. novicida may influence the neutrophil lifespan in an opsonin‐dependent manner. To test this hypothesis, we quantified bacterial uptake, phosphatidylserine (PS) externalization, and changes in nuclear morphology, as well as the kinetics of procaspase‐3, ‐8, and ‐9 processing and activation. To our surprise, we discovered that F. novicida not only failed to accelerate neutrophil death but also diminished and delayed apoptosis in a dose‐dependent, but opsonin‐independent, manner. In keeping with this, studies of conditioned media (CM) showed that neutrophil longevity could be uncoupled from phagocytosis and that F. novicida stimulated neutrophil secretion of CXCL8. Taken together, the results of this study reveal shared and unique aspects of the mechanisms used by Francisella species to manipulate neutrophil lifespan and as such, advance understanding of cell death regulation during infection.
A recent controversial hypothesis suggested that the bactericidal action of antibiotics is due to the generation of endogenous reactive oxygen species (ROS), a process requiring the citric acid cycle ...(tricarboxylic acid TCA cycle). To test this hypothesis, we assessed the ability of oxacillin to induce ROS production and cell death in Staphylococcus epidermidis strain 1457 and an isogenic citric acid cycle mutant. Our results confirm a contributory role for TCA-dependent ROS in enhancing susceptibility of S. epidermidis toward β-lactam antibiotics and also revealed a propensity for clinical isolates to accumulate TCA cycle dysfunctions presumably as a way to tolerate these antibiotics. The increased protection from β-lactam antibiotics could result from pleiotropic effects of a dysfunctional TCA cycle, including increased resistance to oxidative stress, reduced susceptibility to autolysis, and a more positively charged cell surface.
Staphylococcus epidermidis, a normal inhabitant of the human skin microflora, is the most common cause of indwelling medical device infections. In the present study, we analyzed 126 clinical S. epidermidis isolates and discovered that tricarboxylic acid (TCA) cycle dysfunctions are relatively common in the clinical environment. We determined that a dysfunctional TCA cycle enables S. epidermidis to resist oxidative stress and alter its cell surface properties, making it less susceptible to β-lactam antibiotics.
Francisella tularensis infects several cell types including neutrophils, and aberrant neutrophil accumulation contributes to tissue destruction during tularaemia. We demonstrated previously that ...F. tularensis strains Schu S4 and live vaccine strain markedly delay human neutrophil apoptosis and thereby prolong cell lifespan, but the bacterial factors that mediate this aspect of virulence are undefined. Herein, we demonstrate that bacterial conditioned medium (CM) can delay apoptosis in the absence of direct infection. Biochemical analyses show that CM contained F. tularensis surface factors as well as outer membrane components. Our previous studies excluded roles for lipopolysaccharide and capsule in apoptosis inhibition, and current studies of 14C acetate‐labelled bacteria argue against a role for other bacterial lipids in this process. At the same time, studies of isogenic mutants indicate that TolC and virulence factors whose expression requires FevR or MglA were also dispensable, demonstrating that apoptosis inhibition does not require Type I or Type VI secretion. Instead, we identified bacterial lipoproteins (BLPs) as active factors in CM. Additional studies of isolated BLPs demonstrated dose‐dependent neutrophil apoptosis inhibition via a TLR2‐dependent mechanism that is significantly influenced by a common polymorphism, rs5743618, in human TLR1. These data provide fundamental new insight into pathogen manipulation of neutrophil lifespan and BLP function.
Tularemia is a disease characterized by profound neutrophil accumulation and tissue destruction. The causative organism, Francisella tularensis, is a facultative intracellular bacterium that ...replicates in neutrophil cytosol, inhibits caspase activation and profoundly prolongs cell lifespan. Here, we identify unique features of this infection and provide fundamental insight into the mechanisms of apoptosis inhibition. Mitochondria are critical regulators of neutrophil apoptosis. We demonstrate that F. tularensis significantly inhibits Bax translocation and Bid processing during 24-48 h of infection, and in this manner sustains mitochondrial integrity. Downstream of mitochondria, X-linked inhibitor of apoptosis protein (XIAP) and proliferating cell nuclear antigen (PCNA) inhibit caspase-9 and caspase-3 by direct binding. Notably, we find that PCNA disappeared rapidly and selectively from infected cells, thereby demonstrating that it is not essential for neutrophil survival, whereas upregulation of calpastatin correlated with diminished calpain activity and reduced XIAP degradation. In addition, R-roscovitine is a cyclin-dependent kinase inhibitor developed for the treatment of cancer; it also induces neutrophil apoptosis and can promote the resolution of several infectious and inflammatory disorders. We confirm the ability of R-roscovitine to induce neutrophil apoptosis, but also demonstrate that its efficacy is significantly impaired by F. tularensis. Collectively, our findings advance the understanding of neutrophil apoptosis and its capacity to be manipulated by pathogenic bacteria.
Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne’s disease, a chronic intestinal disease of cattle and other ruminants. Diagnostic culture for MAP is typically ...unrewarding until latter stages of the disease. This may be a consequence of oxidative damage to dormant organisms, which results from culture. The purpose of this investigation was to determine the effect of the commercial reducing agent Oxyrase® for Broth (OB) on the recovery of dormant Mycobacterium smegmatis (MS) and MAP. Dormant organisms were inoculated into medium only or that supplemented with media containing serial dilutions of OB. Growth was monitored by optical density for up to 21 days. Treatment of MAP and MS with OB led to significant increases in recovery and growth yield. However, the concentration of OB necessary to promote recovery was dependent on the number of viable organisms present in seed cultures. Mitigating oxidative damage by using OB can facilitate the recovery of dormant mycobacteria. Whether this is a direct or indirect effect has yet to be be established.
Summary
Francisella tularensis
in an intracellular bacterial pathogen that causes a potentially lethal disease called tularemia. Studies performed nearly 100 years ago revealed that neutrophil ...accumulation in infected tissues correlates directly with the extent of necrotic damage during
F
. tularensis
infection. However, the dynamics and details of bacteria–neutrophil interactions have only recently been studied in detail. Herein, we review current understanding regarding the mechanisms that recruit neutrophils to
F
. tularensis
–infected lungs, opsonization and phagocytosis, evasion and inhibition of neutrophil defense mechanisms, as well as the ability of
F
. tularensis
to prolong neutrophil lifespan. In addition, we discuss distinctive features of the bacterium, including its ability to act at a distance to alter overall neutrophil responsiveness to exogenous stimuli, and the evidence which suggests that macrophages and neutrophils play distinct roles in tularemia pathogenesis, such that macrophages are major vehicles for intracellular growth and dissemination, whereas neutrophils drive tissue destruction by dysregulation of the inflammatory response.
Abstract
Background
The highly conserved macromolecular synthesis operon (MMSO) contains both
dnaG
(primase) and
sigA
(primary sigma factor). However, in previously evaluated gram-positive species, ...the MMSO is divergent upstream of
dnaG
. The MMSO of
Bacillus subtilis
contains three open reading frames (ORFs) that are differentially regulated by multiple promoters. In conjunction with studies to determine the expression profile of
dnaG
, the MMSO of
Staphylococus epidermidis
was characterized.
Results
The ORFs of
S. epidermidis
were compared to the previously described MMSO of
B. subtilis
and two additional ORFs in
S. epidermidis, serp1129
and
serp1130
, were identified. The largest transcript, 4.8 kb in length, was expressed only in exponential growth and encompassed all four ORFs (
serp1130, serp1129, dnaG
, and
sigA
). A separate transcript (1.5 kb) comprising
serp1130
and
serp1129
was expressed in early exponential growth. Two smaller transcripts 1.3 and 1.2 kb in size were detected with a
sigA probe
in both exponential and post-exponential phases of growth. Western blot analysis correlated with the transcriptional profile and demonstrated that Serp1129 was detected only in the exponential phase of growth. Computational analysis identified that Serp1130 contained a CBS motif whereas Serp1129 contained an ATP/GTP binding motif. Functional studies of Serp1129 demonstrated that it was capable of binding both ATP and GTP. Comparisons with a
sigB:dhfr
mutant revealed that the 1.3 kb
sigA
transcript was regulated by a σ
B
-dependent promoter.
Conclusions
These studies demonstrated that the
S. epidermidis
1457 MMSO contains two ORFs (
serp1129
and
serp1130
) not described within the
B. subtilis
MMSO and at least three promoters, one of which is σ
β
-dependent. The transcriptional regulation of
sigA
by σ
B
provides evidence that the staphylococcal σ
B
-dependent response is controlled at both the transcriptional and post-transcriptional level. The conservation of
serp1129
across multiple gram-positive organisms and its capability to bind ATP and GTP support the need for further investigation of its role in bacterial growth.
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